Lipid bilayers are aggregates of lipids held together by hydrophobic interactions that form a variety of different structures, such as closed spherical vesicles, planar sheets, flat discs, globules, tubes, and helices (Kunitake, T. Angew. Chem., Int. Ed. Engl., 31:709 (1992)). Such lipid bilayer assemblies form spontaneously from a variety of lipids and have found use in a wide variety of technical fields. For example, planar lipid bilayer assemblies are used as optical sensors for the detection of target analytes (U.S. Pat. No. 5,616,790), as unique separation materials for peptides, proteins, nucleotides, and oligonucleotides i.e., immobilized artificial membranes (Pidgeon, C. et al., Anal. Biochem., 176:36 (1989)), as biosensors (See H. T. Tien et al. in “Molecular Electronics: Biosensors and Biocomputers”, ed. F. T. Hong, Plenum Press, New York (1989) at pages 259-268), and as planar bilayer lipid membranes for studying ligand-receptor interactions at the lipid-water interface (WO 98/23948; WO 01/26800; U.S. Pat. No. 5,922,594). Spherical bilayer lipid particles, such as liposomes, have also found use as carriers of drugs or diagnostic agents.
Often it is desirable to attach or immobilize a test material or a reaction component to the lipid bilayer. For example, many types of analytical chemistry techniques rely on the immobilization of one or more of components of a reaction. Immunoassay techniques typically involve immobilizing one of the components to the lipid surface. When the test sample is washed across the lipid surface, components in the test sample having binding affinity to the immobilized component are captured for detection.
In the case of closed spherical lipid vesicles, such as micelles and liposomes, molecules are often attached to the outer vesicle surface for a variety of purposes. For example, the attached molecule can be a therapeutic agent, and attaching the agent to the lipid bilayer serves to alter the pharmacokinetics of the agent (see, for example, U.S. Pat. No. 6,326,353). The attached molecule can be a targeting moiety that serves to direct or ‘target’ the vesicle to a desired site after in vivo administration (see, for example, Allen. T. M., et al., Biochim. Biophys. Acta, 1237:99-108 (1995); Blume, G., et al., Biochim. Biophys. Acta, 1149:180-184 (1993); U.S. Pat. Nos. 6,316,024; 6,214,388). Typically, the ligand is attached to the liposome surface through a linker, often a polymer chain.
Lipid vesicles having an attached ligand are typically prepared by one of several techniques. One approach involves preparation of lipid vesicles which include an end-functionalized lipid-polymer derivative; that is, a lipid-polymer conjugate where the free polymer end is reactive or “activated” (see, for example Zalipsky et al., Bioconjugate Chem., 4:296 (1993); Zalipsky et al., FEBS Letters, 353:71 (1994); Zalipsky et al., J. Control. Rel., 39:153 (1996); Zalipsky et al., Bioconjugate Chem., 8(2): 111 (1997)). The lipid-activated polymer conjugate is included in the lipid mixture during liposome formation. After liposome formation, the activated polymer ends are reacted with the desired ligand (Zalipsky et al., Bioconjugate Chem., 4:296 (1993)). The disadvantage to this approach is the difficulty in reacting all of the activated ends with a ligand. The approach also requires a subsequent step for separation of the unreacted ligand from the liposome composition.
In another approach, a lipid-polymer-ligand conjugate is prepared and is included in the lipid composition at the time of liposome formation (Zalipsky et al., Bioconjugate Chem., 8(2):111 (1997)). This approach suffers from the disadvantage that some of the valuable ligand faces the inner aqueous compartment of the liposome and is unavailable for interaction with the intended target.
In another approach, a suspension of liposomes is incubated with a micellar suspension of lipid-polymer-ligand conjugates to achieve insertion of the conjugates into the liposomes' bilayers (see, for example, Uster et al., FEBS Letters, 386:243 (1996); Zalipsky et al., Bioconjugate Chem., 8(2):111 (1997); U.S. Pat. Nos. 5,891,468, 6,316,024). This approach results in successful insertion of the conjugate, provided the incubation conditions are suitable and sufficient time is permitted. In some cases, the long incubation time or the required temperature to achieve insertion leads to inactivation of the ligand and/or release of liposomal contents.
Thus, there remains a need in the art for a method of attaching a desired ligand to a preformed lipid assembly.
The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and a study of the drawings.